This invention relates to bacterial diagnostics, and more particularly to detection of xcex2-hemolytic Group A Streptococcus (GAS).
Streptococcus pyogenes is a group A streptococcal gram-positive bacterium that is the etiological agent of several diseases in humans, including pharyngitis and/or tonsillitis, skin infections (impetigo, erysipelas, and other forms of pyoderma), acute rheumatic fever (ARF), scarlet fever (SF), poststreptococcal glomerulonephritis (PSGN), and a toxic shock-like syndrome (TSLS). On a global basis, ARF is the most common cause of pediatric heart disease. For example, it is estimated that in India, more than six million school-aged children suffer from rheumatic heart disease. In the United States, xe2x80x9csore throatxe2x80x9d is the third most common reason for physician office visits and S. pyogenes is recovered from about 30% of children with this complaint. There are about 25-35 million cases of streptococcal pharyngitis per year in the United States, responsible for about 1-2 billion dollars per year in health care costs.
The invention provides for methods of identifying group A streptococcus (GAS) in a biological sample. Primers and probes for detecting GAS are provided by the invention, as are kits containing such primers and probes. Methods of the invention can be used to rapidly identify GAS nucleic acids from specimens for diagnosis of GAS infection. Using specific primers and probes, the methods include amplifying and monitoring the development of specific amplification products using real-time PCR.
In one aspect, the invention features a method for detecting the presence or absence of Group A Streptococcus (GAS) in a biological sample from an individual. The method to detect GAS includes performing at least one cycling step, which includes an amplifying step and a hybridizing ste. The amplifying step includes contacting the sample with a pair of ptsI primers to produce a ptsI amplification product if a GAS ptsI nucleic acid molecule is present in the sample, and the hybridizing step includes contacting the sample with a pair of ptsI probes. Generally, the members of the pair of ptsI probes hybridize to the amplification product within no more than five nucleotides of each other. A first ptsI probe of the pair of ptsI probes is typically labeled with a donor fluorescent moiety and a second ptsI probe of the pair of ptsI probes is typically labeled with a corresponding acceptor fluorescent moiety. The method further includes detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety of the first ptsI probe and the acceptor fluorescent moiety of the second ptsI probe. The presence of FRET is usually indicative of the presence of GAS in the biological sample, and the absence of FRET is usually indicative of the absence of GAS in the biological sample. The method can still further include determining the melting temperature between one or both of the ptsI probe(s) and the ptsI amplification product. The melting temperature can confirm the presence or the absence of the GAS.
A pair of ptsI primers generally includes a first ptsI primer and a second ptsI primer. The first ptsI primer can include the sequence 5xe2x80x2-AAA TGC AGT AGA AAG CTT AGG-3xe2x80x2 (SEQ ID NO:1), and the second ptsl primer can include the sequence 5xe2x80x2-TGC ATG TAT GGG TTA TCT TCC-3xe2x80x2 (SEQ ID NO:2). The first ptsI probe can include the sequence 5xe2x80x2-TTG CTG ATC CAG AAA TGA T-3xe2x80x2 (SEQ ID NO:3), and the second ptsI probe can include the sequence 5xe2x80x2-AGC CAG GTT AAA GAA ACG ATT CGC-3xe2x80x2 (SEQ ID NO:4).
The members of the pair of ptsI probes can hybridize within no more than two nucleotides of each other, or can hybridize within no more than one nucleotide of each other. A representative donor fluorescent moiety is fluorescein, and representative acceptor fluorescent moiety is selected from the group consisting of LC(trademark)-RED 640 (LightCycler(trademark)-Red 640-N-hydroxysuccinimide ester), LC(trademark)-RED 705 (LightCycler(trademark)-Red 705-Phosphoramidite), and cyanine dyes such as CY5 and CY5.5.
In one aspect, the detecting step includes exciting the biological sample at a wavelength absorbed by the donor fluorescent moiety and visualizing and/or measuring the wavelength emitted by the acceptor fluorescent moiety. In another aspect, the detecting comprises quantitating the FRET. In yet another aspect, the detecting step is performed after each cycling step, and further, can be performed in real-time.
Generally, the presence of the FRET within 50 cycles, or within 40 cycles, or within 30 cycles, indicates the presence of a GAS infection in the individual. Representative biological samples include throat swabs, tissues and bodily fluids.
The above-described methods can further include preventing amplification of a contaminant nucleic acid. Preventing amplification can include performing the amplification step in the presence of uracil and treating the biological sample with uracil-DNA glycosylase prior to a first amplification step. In addition, the ycling step can be performed on a control sample. A control sample can include the GAS ptsI nucleic acid molecule. Alternatively, such a control sample can be amplified using a pair of control primers and hybridized using a pair of control probes. The control primers and the control probes are usually other than the ptsI primers and the ptsI probes, respectively. A control amplification product is produced if control template is present in the sample, and the control probes hybridize to the control amplification product.
In another aspect of the invention, there are provided articles of manufacture, including a pair of ptsI primers; a pair of ptsI probes; and a donor fluorescent moiety and a corresponding fluorescent moiety. A pair of ptsI primers generally includes a first ptsI primer and a second ptsI primer. A first ptsI primer can include the sequence 5xe2x80x2-AAA TGC AGT AGA AAG CTT AGG-3xe2x80x2 (SEQ ID NO:1), and the second ptsI primer can include the sequence 5xe2x80x2-TGC ATG TAT GGG TTA TCT TCC-3xe2x80x2 (SEQ ID NO:2). A pair of ptsI probes can include a first ptsI probe and a second ptsI probe. A first ptsI probe can include the sequence 5xe2x80x2-TTG CTG ATC CAG AAA TGA T-3xe2x80x2 (SEQ ID NO:3), and the second ptsI probe can include the sequence 5xe2x80x2-AGC CAG GTT AAA GAA ACG ATT CGC-3xe2x80x2 (SEQ ID NO:4). The probes in such articles of manufacture can be labeled with a donor fluorescent moiety and with a corresponding acceptor fluorescent moiety. The articles of manufacture also can include a package label or package insert having instructions thereon for using the pair of ptsI primers and the pair of ptsI probes to detect the presence or absence of GAS in a biological sample.
In yet another aspect, the invention provides a method for detecting the presence or absence of GAS in a biological sample from an individual. Such a method includes performing at least one cycling step, wherein a cycling step comprises an amplifying step and a hybridizing step. An amplifying step includes contacting the sample with a pair of ptsI primers to produce a ptsI amplification product if a GAS ptsI nucleic acid molecule is present in the sample. A hybridizing step includes contacting the sample with a ptsI probe, wherein the ptsI probe is labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety. The method further includes detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor fluorescent moiety of the ptsI probe. The presence or absence of FRET is indicative of the presence or absence of GAS in the sample. Amplification can employ a polymerase enzyme having 5xe2x80x2 to 3xe2x80x2 exonuclease activity, and the donor and acceptor fluorescent moieties can be within no more than 5 nucleotides of each other on the probe. In such a method, the ptsI probe can include a nucleic acid sequence that permits secondary structure formation that results in spatial proximity between the donor and the acceptor fluorescent moiety. In the above-described methods, the acceptor fluorescent moiety can be a quencher.
In another aspect, the invention provides a method for detecting the presence or absence of GAS in a biological sample from an individual. Such a method includes performing at least one cycling step, wherein a cycling step comprises an amplifying step and a dye-binding step. An amplifying step includes contacting the sample with a pair of ptsI primers to produce a ptsI amplification product if a GAS ptsI nucleic acid molecule is present in the sample. A dye-binding step comprises contacting the ptsI amplification product with a nucleic acid binding dye. The method further includes detecting the presence or absence of binding of the nucleic acid binding dye to the amplification product. The presence of binding is usually indicative of the presence of GAS in the sample, and the absence of binding is usually indicative of the absence of GAS in the sample. Representative nucleic acid binding dyes include SYBRGREENI(copyright), SYBRGOLD(copyright), and ethidium bromide. Such a method can further include determining the melting temperature between the ptsI amplification product and the nucleic acid binding dye. The melting temperature can confirm the presence or absence of the GAS.
Unless othehrwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.